The Effects of Peripubertal THC Exposure in Neurodevelopmental Rat Models of Psychopathology

Adolescent exposure to cannabinoids as a postnatal environmental insult may increase the risk of psychosis in subjects exposed to perinatal insult, as suggested by the two-hit hypothesis of schizophrenia. Here, we hypothesized that peripubertal Δ9-tetrahydrocannabinol (aTHC) may affect the impact of prenatal methylazoxymethanol acetate (MAM) or perinatal THC (pTHC) exposure in adult rats. We found that MAM and pTHC-exposed rats, when compared to the control group (CNT), were characterized by adult phenotype relevant to schizophrenia, including social withdrawal and cognitive impairment, as revealed by social interaction test and novel object recognition test, respectively. At the molecular level, we observed an increase in cannabinoid CB1 receptor (Cnr1) and/or dopamine D2/D3 receptor (Drd2, Drd3) gene expression in the prefrontal cortex of adult MAM or pTHC-exposed rats, which we attributed to changes in DNA methylation at key regulatory gene regions. Interestingly, aTHC treatment significantly impaired social behavior, but not cognitive performance in CNT groups. In pTHC rats, aTHC did not exacerbate the altered phenotype nor dopaminergic signaling, while it reversed cognitive deficit in MAM rats by modulating Drd2 and Drd3 gene expression. In conclusion, our results suggest that the effects of peripubertal THC exposure may depend on individual differences related to dopaminergic neurotransmission.


Introduction
It is well accepted that exposure of the immature brain to several insults (such as prenatal infection, drugs of abuse, environmental pollutants, etc.) combined with genetic risk factors could lead to aberrant neurodevelopment, emerging psychopathology in adulthood including schizophrenia (SCZ), autism or obsessive-compulsive disorders [1][2][3][4][5]. Cannabis is the most common illicit drug used during pregnancy in Western countries, and its use is constantly increased in recent decades, irrespective of its detrimental health effects, which have long been documented [6]. Brain development begins in utero and extends through late adolescence [7], when it undergoes remodeling in the form of synaptic pruning, myelinization, and changes in neurotransmitter systems [8][9][10]. Indeed, prenatal age and adolescence are both crucial periods when exposure to drug abuse affects the central nervous system (CNS) development, leading to cognitive, emotional, and social alterations in adulthood, which are common hallmarks of psychopathology [11]. This is the cornerstone of the two-hit hypothesis of SCZ, stating that genetic and/or environmental factors might alter early CNS development producing long-term vulnerability to a "second hit" which would lead to the onset of SCZ symptoms. Therefore, gestational and/or adolescent exposure to ∆ 9 -tetrahydrocannabinol (THC), the main psychoactive component of Cannabis sativa, might alter the normal activity of the endocannabinoid system (ECS) due to its high affinity for the cannabinoid CB1 receptor (Cnr1) gene [12,13], the overstimulation of which contribute to dysregulate the neurodevelopmental processes leading to neuroanatomical and functional alterations during neurogenesis [6,14]. A functional interaction between the dopaminergic (DAergic) system and the ECS due to the coexpression of Cnr1 and dopamine D2 receptors (Drd2) genes [15] and to their signal transduction convergence [16] has been suggested both at the level of the prefrontal cortex (PFC) and in several limbic structures as possible neuroanatomical loci for neurodevelopmental disorders [11]. Thus, it appears to be likely that altered DAergic neurotransmission at the PFC level may also play a role in the long-lasting detrimental effects induced by perinatal insult per se or combined with peripubertal THC (aTHC) exposure. However, whether the potential effects of aTHC exposure as a "second hit" may be specifically related to the kind of pre/perinatal insult as a "first hit" is still debated.
Based on this background, the present study aimed to investigate if aTHC exposure could modify the effects of pre/perinatal insult induced by gestational methylazoxymethanol acetate (MAM) or perinatal THC (pTHC) exposure, two well-validated neurodevelopmental models of psychopathology [13,17,18], which are characterized by behavioral and molecular alterations similar to those described in subjects with neurodevelopmental disorders, further supporting their translational value [13,19]. More specifically, in adulthood, we assessed whether the cognitive deficits and social impairment typically described in the pTHC and MAM offspring and usually paralleled by changes in the ECS and/or DAergic system at the PFC level [13,17], might be modified by aTHC exposure (Figure 1).  19 to PND 39, resulting male offspring were i.p. treated twice/day with increasing doses of THC (aTHC) or respective vehicle (aVEH). Behavioral tests (open field test, novel object recognition test and social interaction test) were conducted in adulthood from PND 100. After completion, the neurochemical analyses (quantitative real-time PCR and DNA methylation by pyrosequencing) were performed in the prefrontal cortex of rats. THC model: Pregnant rats were treated with THC (pTHC, 5 mg/kg/day; per os) or vehicle (CNT; 1 mL/kg/day; per os) from gestational day (GD) 15 to postnatal day (PND) 9. From PND 19 to PND 39, the resulting male offspring were i.p. treated twice/day with increasing doses of THC (aTHC) or respective vehicle (aVEH). Behavioral tests (open field test, novel object recognition test and social interaction test) were conducted in adulthood from PND 100. After completion, the neurochemical analyses (quantitative real-time PCR and DNA methylation by pyrosequencing) were performed in the prefrontal cortex of rats.  (Figure 2A,B).

Peripubertal THC Exposure in Prenatally MAM-Exposed Rats
As shown in Figure 2C (Figure 2A,B).
The effect of aTHC exposure alone or combined with pTHC on mRNA expression of dopamine D2 receptors in PFC is depicted in Figure 5D

Discussion
The findings of the present study confirm that pre/perinatal insult in rats evoked by maternal MAM or THC exposure causes harmful effects in offspring, namely a) SCZ-like phenotype, b) altered DAergic and/or cannabinoid neurotransmission, in a manner similar to that previously described both in preclinical studies and in SCZ subjects, thus further supporting the translational value of these two models of psychopathology [13,14,[19][20][21][22]. More specifically, MAM or pTHC adult rats showed social and cognitive deficits, as described by the reduced time of interaction (as an index of social withdrawal) in the social interaction test and impaired short-term recognition memory in the novel object recognition test, which are often considered to be SCZ-like symptoms [11,23]. Furthermore, no difference was found in the number of interactions, as well as no spontaneous locomotor hyperactivity was described, in agreement with previous results [14]. The locomotor performance paradigms served as an internal control for possible unspecific stimulant effects; thus, our study reinforces the original findings that cognitive impairment in the novel object recognition test and social deficit in the social interaction

Discussion
The findings of the present study confirm that pre/perinatal insult in rats evoked by maternal MAM or THC exposure causes harmful effects in offspring, namely (a) SCZ-like phenotype, (b) altered DAergic and/or cannabinoid neurotransmission, in a manner similar to that previously described both in preclinical studies and in SCZ subjects, thus further supporting the translational value of these two models of psychopathology [13,14,[19][20][21][22]. More specifically, MAM or pTHC adult rats showed social and cognitive deficits, as described by the reduced time of interaction (as an index of social withdrawal) in the social interaction test and impaired short-term recognition memory in the novel object recognition test, which are often considered to be SCZ-like symptoms [11,23]. Furthermore, no difference was found in the number of interactions, as well as no spontaneous locomotor hyperactivity was described, in agreement with previous results [14]. The locomotor performance paradigms served as an internal control for possible unspecific stimulant effects; thus, our study reinforces the original findings that cognitive impairment in the novel object recognition test and social deficit in the social interaction test are robust phenotypes in pTHC or MAM rats, as valuable models of neurodevelopmental disorders.
MAM or pTHC exposure also induced alterations in the transcriptional regulation of the genes encoding for cannabinoid CB1 (Cnr1), dopamine D2 (Drd2) or D3 (Drd3) receptors at the level of PFC of adult rats, further confirming the hypothesis of DA-cannabinoid interaction as a molecular substrate of neurodevelopmental disorders [24]. However, MAM or pTHC exposure differentially affected the DNA methylation at Cnr1 or Drd2 gene regulatory regions, which were not always followed by significant alterations in gene expression. Thus, these findings suggest that alterations in dopamine D2 and cannabinoid CB1 mRNA levels evoked by pre/perinatal insults, which seem to be involved in the altered phenotype of adult rats, might also be regulated by other mechanisms at epigenetic levels, such as histone modifications, or most likely at post-transcriptional levels, e.g., via microRNAs [25]. Further investigations are indeed warranted to clarify these issues. We also observed a significant correlation between Drd2, but not Cnr1 receptor gene expression and cognitive impairment in MAM rats in agreement with the hypothesis that altered DAergic neurotransmission at the PFC level may play a role in the cognitive deficits of SCZ as a neurodevelopmental disorder [26]. We cannot exclude that the hippocampus may also play a role in the cognitive performance of MAM or pTHC rats, since it is becoming increasingly clear that neurodevelopmental disorders are not only due to a circumscribed deficit in the PFC and/or hippocampus, but also represent a distributed impairment involving hippocampal-PFC connectivity [27]. However, in post-mortem studies, contradictory results have been found since decreased [23,[28][29][30], increased [31], or unchanged [30,32] cannabinoid CB1 and/or dopamine D2 receptor brain expression has been detected. Conflicting data may be due to differences in patient symptom severity, amount of THC exposure, co-pharmacological treatments, or diagnostic methods in studies.
In addition to the well-documented detrimental effects of perinatal THC exposure, Cannabis use during adolescence is often associated with an increased risk of developing neuropsychiatric disorders in later life, due to changes at the level of GABAergic, glutamatergic, serotonergic, DAergic neurotransmission, and/or endocannabinoid signaling [6,33]. Evidence from animal studies consistently indicates that adolescent treatment with cannabinoid agonists induces lasting behavioral and molecular changes in adulthood, mimicking psychopathology [14]. In our experimental conditions, aTHC exposure significantly affected social activity, while it failed to induce a significant cognitive impairment in adult rats, partially in line with previous observations [6,14]. These discrepancies may be due to several factors, such as heterogeneity in Cannabis types (phytocannabinoid "THC" vs. synthetic cannabinoids "HU-210", "CP-55940", "WIN55,212-2"), doses, and timing of treatment (peripubertal vs. postpubertal), as well as to gender difference and to different cognitive tasks. At the PFC level, neither the cannabinoid CB1 nor the dopamine D2/D3 receptor gene expression was affected, suggesting that aTHC exposure may lead to social withdrawal by the involvement of different neurotransmitter systems, as previously suggested [14,33].
Based on the two-hit hypothesis, it has been suggested that SCZ, as a neurodevelopmental disorder, may be related to the combination of early prenatal and, likely environmental, postnatal insult. Consistently, combining exposure to prenatal insults and peripubertal stress in rodents may induce synergistic detrimental effects in adulthood [34]. Indeed, Cannabis exposure during adolescence which, per se, seems to be associated with an increased risk of developing psychopathology in adulthood [14], may induce more pronounced behavioral and molecular alterations in pre/perinatal stressed rodents. In pTHC rats, aTHC exposure did not further impair the social and cognitive deficits induced by pTHC exposure, suggesting that pTHC exposure at the dose of 5 mg/kg may induce strong detrimental effects, which cannot be further exacerbated by aTHC insult. At the molecular level, aTHC exposure significantly reversed the cannabinoid CB1 but not the dopamine D2 mRNA receptor overexpression induced by pTHC, suggesting a different impact of aTHC exposure on cannabinoid/DAergic system alteration induced by pTHC. Interestingly, aTHC exposure further reduced the dopamine D2 DNA methylation in pTHC, suggesting that THC exposure in adolescence may differentially impact epigenetic marks based on prenatal insult. As also mentioned above, we cannot exclude that another epigenetic mechanism, such as histone modifications and/or microRNAs, may also be involved in the effects elicited by aTHC exposure [25]. Overall, these findings indicate that aTHC exposure seems to solely revert the cannabinoid CB1 gene overexpression in the PFC of pTHC rats, whose reduction, in turn, does not impact the behavioral performances of pTHC/aTHC rats. Similarly, in MAM offspring, aTHC insult did not induce additive or synergistic detrimental effects at the level of social behavior. Although it may reflect a ceiling effect, the social withdrawal elicited by the aTHC exposure may be related to the involvement of different neurotransmitter systems, as recently suggested [14,25].
Contrary to our expectation, aTHC exposure improved the cognitive deficit of MAM rats in the novel object recognition test. How aTHC might reverse the cognitive impairment remains to be fully clarified. Based on the molecular findings, we assume that it may be due to the reduced dopamine D2/D3 receptor mRNA overexpression in the PFC of MAM rats, in agreement with previous studies showing that altered dopamine D2/D3 receptor expression may be involved in the cognitive deficits of neurodevelopmental disorders [13,21]. Notably, contrary to the pTHC model, aTHC did not revert the CB1 overexpression; thus, we could just speculate that in the neurodevelopmental MAM model, a pivotal role in the better cognitive performance induced by aTHC may be related to the changes in dopamine D2/D3 receptors more than the cannabinoid CB1 receptors. Further studies should clarify the involvement of different DAergic neurotransmission elements, such as dopamine D1-like receptors or dopamine transporter, both in the altered phenotype induced by pre/perinatal insults and in response to adolescent Cannabis exposure. To the best of our knowledge, this is the first study carried out in the MAM model of SCZ with peripubertal exposure to phytocannabinoid THC and not synthetic cannabinoids [35]. Specifically, adolescent exposure to the synthetic cannabinoid WIN55,212-2 attenuated the enhanced locomotor response to amphetamine in MAM rats, without impacting DA neuron activity at the level of the ventral tegmental area [35]. On the other hand, Aguilar et al., 2018 [36] showed that adolescent exposure to synthetic cannabinoids such as the CB1 agonist WIN55,212-2 or the FAAH inhibitor URB597 significantly increased the proportion of susceptible rats that developed an SCZ-like hyper-DAergic phenotype. Interestingly, our results are also in agreement with Lecca et al. (2019) [37], showing that adolescent THC intake attenuates the disruption of DAergic signaling in prenatal Poly I:C-exposed rats, a well-validated neurodevelopmental model of SCZ based on maternal immune activation [11]. The discrepancies with our results may be due to the different experimental designs, such as the pharmacology of the cannabinoids used (phytocannabinoid vs. synthetic; full vs. partial agonist; direct vs. indirect agonist), the adolescent cannabinoid protocol treatment (11 vs. 25 days), the generation of MAM offspring (first vs. second), and the brain region (PFC vs. ventral tegmental area).
In conclusion, our data confirm that different gestational insults such as MAM or THC exposure may lead at least to alteration of the DAergic (in terms of dopamine D2/D3 receptor expression) and endocannabinoid signaling (through the cannabinoid CB1 receptor altered expression), similarly to the changes previously described in SCZ subjects [13,19], further supporting the translational value of gestational MAM or pTHC exposure as neurodevelopmental animal models (Table 1). Although we just limited our study to assess the cannabinoid CB1 and dopamine D2/D3 receptor, we cannot exclude that the altered phenotype of MAM or pTHC rats, as neurodevelopmental animal models, may also be due to neuroimmune dysfunctions, as previously suggested [2][3][4][5]. On the contrary, the impact of aTHC exposure per se or combined with pre/perinatal insults is more complex than expected, and the combination of gestational and postnatal insults, as the main mechanism of the two-hit hypothesis of SCZ [34], needs to be further assessed. In this scenario, behavioral tasks for evaluating different cognitive and social aspects, as well as molecular analysis for assessing further potential neurochemical anomalies (i.e., immune cell dysfunction), should be helpful to better understand the effects of pre/perinatal insult alone or combined with postnatal insult. Table 1. Summary of the behavioral and molecular effects of peripubertal treatment with THC (aTHC) or vehicle (aVEH) in MAM or perinatally THC (pTHC)-exposed rats in adulthood.

Animals
Pregnant Sprague Dawley rats were obtained from Charles River (Germany) at gestational day (GD) 13 and housed individually. Environmental conditions during the whole study were constant: relative humidity 50-60%, temperature 23 • C ± 1 • C, normal 12 h light-dark cycle (light on 7 a.m. to 7 p.m.). Food and water were available ad libitum. All procedures were performed in accordance with EU Directive No. 2010/63/EU and approved by the Animal Care Committee of the Faculty of Medicine, Masaryk University, Czech Republic, and the Czech Governmental Animal Care Committee, in compliance with Czech Animal Protection Act No. 246/1992.

Drugs and Experimental Design
Methylazoxymethanol acetate (MAM; Midwest Research Institute, Kansas City, MO, USA) was dissolved in saline and administered intraperitoneally (i.p.) at dose of 22 mg/kg in 1 mL/kg volume on GD 17 with saline solution as a control (CNT), as previously described [17,18,[38][39][40][41] (Figure 1, MAM model. THC (10 mg/mL in ethanol solution) obtained from the University of Chemistry and Technology, Prague, was prepared as previously described [42,43] (Figure 1, THC model). For perinatal administration, pregnant rats received daily oral gavage of THC (pTHC, 5 mg/kg/day) or sesame oil in control group (CNT) from GD 15 to postnatal day (PND) 9 [13]. The administered dose is equivalent to the current estimates of moderate exposure to THC in humans, correcting for differences in the route of administration and the body surface area [44]. No cross-fostering was used; the mothers were regularly weighed, and no differences in the body weight gains were observed among THC, MAM, or CNT-treated dams. The offspring were weaned on PND 22 and housed in groups of 2-3. For the peripubertal administration, THC was dissolved in vehicle solution (1% ethanol, 2% Tween 80, and saline). From PND 29 to PND 39, different groups of male CNT-, pTHC-or MAM-exposed rats (n = 12-15) were treated twice/day with increasing doses of THC (aTHC) or vehicle (aVEH), according to the following treatment schedule: 2.5 mg/kg/day (PND 29-31, i.p.), 5 mg/kg/day (PND 32-35, i.p.) and 10 mg/kg/day (PND 36-39, i.p.) as previously described [45]. Male rats were randomly assigned to the experimental procedures, and care was taken to avoid assigning more than two animals from the same litter to the same experimental group.
The drug treatment period in rats was carried out at the equivalent time of the childhood/periadolescent phase in humans [10], and it was also based on previous results both in MAM and pTHC [13,20] models. The adult (from PND 100) rats were subjected to behavioral tests with 5 days in between two consecutive tests, as previously described [46][47][48][49]. Immediately thereafter, they were decapitated in short ether anesthesia, and their brains were harvested. The PFC (corresponding to an area that included the rostral pole of the brain and delimited medially by the interhemispheric fissure, laterally by the corpus callosum, and caudally extended to AP + 2.7, according to Paxinos and Watson (1998) [50]) was dissected on ice by hand under microscopic control within 2 min, immediately frozen on liquid nitrogen, and stored at −80 • C until analysis.

Spontaneous Locomotor Activity in the Open Field Test (OFT)
The exploratory activity was assessed in moderately illuminated (80 lx) cubic metal arena (60 × 60 × 60 cm), as previously described [51][52][53][54][55]. Rats were placed gently in the center of the arena and allowed to explore. The vertical (number of rearing episodes) and the horizontal (number of squares crossed with all paws) exploratory activity was counted in 30 min sessions, recorded, and then scored offline by 2 observers blinded to the treatment groups. The arena was cleaned with 0.1% acetic acid and dried after each trial.

Social Interaction (SI) Test
The test was carried out in a moderately illuminated room (120 lx), as previously described [17,43,46,47]. Each animal was allowed to freely explore an unfamiliar congener in a metal arena (60 × 60 × 60 cm) for 10 min. The arena was cleaned with 0.1% acetic acid and dried after each trial. Social behaviors were defined as sniffing, following, grooming, mounting, and nosing. The whole testing phase was recorded and analyzed by two observers blinded to the treatment groups. The time spent on social behaviors and the number of interactions were evaluated.

Novel Object Recognition (NOR) Test
The experimental apparatus used for the NOR test was an arena (43 × 43 × 32 cm) made of plexiglass situated in a moderately illuminated room (120 lx). Each animal was placed in the arena and allowed to explore two identical, previously unseen objects for 5 min (familiarization phase). After an inter-trial interval of 3 min, one of the two familiar objects was replaced by a novel, previously unseen object, and rats were returned to the arena for the 5 min test phase [13,17,20]. Each phase was recorded and analyzed separately by two observers blinded to the treatment groups. The time spent exploring the familiar object (Tf) and the novel object (Tn) were scored. The discrimination index (DI) was calculated as DI = (Tn − Tf)/(Tn + Tf). The arena and all objects were cleaned with 0.1% acetic acid and dried after each trial.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated from rat dissected brain areas using TRIzol ® Reagent (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's protocol. The concentration of each purified RNA was detected by NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The ratio of optical density at 260 and 280 nm was used to assess protein contamination: a value of 1.8-2.1 was considered acceptable. Starting with 0.5 µg of total RNA, complementary DNA (cDNA) was obtained with a mix of random hexamers and oligo-dT primers for mRNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). Diluted cDNAs were then used to assess the relative abundance of each mRNA species by qRT-PCR, using the SensiFAST TM SYBR ® Lo-ROX Kit (Bioline, London, UK) on a 7500 Fast Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Each reaction mix was prepared with 2 µL of diluted cDNA, 7.5 µL of SensiFAST SYBR, and 10 pmol of each primer with a final volume of 15 µL. Sequences of the primers used for the amplification are reported in Supplementary Table S1. To provide an accurate quantification of the initial target in each PCR reaction, the amplification plot was examined, and the point of early log phase of product accumulation was defined by assigning a fluorescence threshold above background, defined as the threshold cycle number or Ct, as previously described [19]. Differences in threshold cycle number were used to quantify the relative amount of the PCR targets contained within each tube. After PCR, a dissociation curve (melting curve) was constructed in the range of 60 to 95 • C [56] to evaluate the specificity of the amplification products. The relative expression of different amplicons was calculated by the Delta-Delta Ct (∆∆CT) method and converted to 2 −∆∆Ct for statistical analysis [57]. All data were normalized to two endogenous reference genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin (β-ACT).

DNA Methylation Analysis by Pyrosequencing
Methylation status of Cnr1 and Drd2 genes was determined using pyrosequencing of bisulfite-converted DNA [13,19,20]. After extraction, DNA concentrations were estimated by measuring the absorbance at 260 nm, whereas sample purities were by considering the ratios of the absorbance values of 260 vs. 280 nm (A260/280 = 1.8). Each purified DNA was subjected to bisulfite modification by means of the EZ DNA Methylation-Gold TM Kit (Zymo Research, Orange, CA, USA), according to the manufacturer's protocol. DNA methylation status at each CpG site under study in the regulatory region of the genes (see Supplementary Figure S2 for gene details) was then assessed using a pyrosequencing assay. Bisulfite-treated DNA was first amplified by PyroMark PCR Kit (Qiagen, Hilden, Germany) with a biotin-labeled primer according to the manufacturer's recommendations. PCR conditions were as follows: 95 • C for 15 min, followed by 45 cycles of 94 • C for 30 s, 56 • C for 30 s, 72 • C for 30 s, and, finally, 72 • C for 10 min. PCR products were then verified by agarose electrophoresis, immobilized to Streptavidin Sepharose High-Performance (GE Healthcare, Chicago, IL, USA) beads via biotin affinity, and denatured to allow the annealing with the sequencing primers. The sequencing was performed on a PyroMark Q24 ID using Pyro Mark Gold reagents (Qiagen). Rat Cnr1 primers for PCR amplification and sequencing were generated according to PyroMark Assay Design software version 2.0 (Qiagen, Hilden, Germany) to analyze 4 CpG sites within the gene regulatory region. A specific PyroMark CpG assay (Qiagen, Hilden, Germany) was instead used to analyze the rat Drd2 regulatory region, targeting 6 CpG sites. Details of the sequences under study and primers and assays employed are shown in Supplementary Table S2. Methylation's level was analyzed using the PyroMark Q24 ID version 1.0.9 software, which calculates the methylation percentage mC/(mC + C) (mC = methylated cytosine, C = unmethylated cytosine) for each CpG site, allowing quantitative comparisons. Quantitative methylation results were expressed as the average methylation percentage of all the investigated CpG sites.

Statistical Analysis
The results are presented as group mean ± SEM. Behavioral data were first tested for normality distribution using the Shapiro-Wilk test. As normality tests have little power to detect non-Gaussian distributions with small data sets, we did not explicitly test for the normality of our biochemical data sets. Data were analyzed using two-way ANOVA (factor 1: model "MAM or pTHC"; factor 2: peripubertal THC treatment "aTHC") followed by Tukey's post hoc if appropriate. Unpaired t-test was used to analyze independent data. For correlation analysis, Spearman's coefficient was used. Statistical evaluations were performed using specialized software (Graph-Pad Prism 9.0). A p level of 0.05 or less was considered indicative of a significant difference.